The receptor tyrosine kinase (RTK) family includes receptors that are crucial for the growth and differentiation of a variety of cell types (Yarden and Ullrich, Ann. Rev. Biochem. 57:433–478, 1988; Ullrich and Schlessinger, Cell 61:243–254, 1990). Several receptor tyrosine kinases, and growth factors that bind thereto, have been suggested to play a role in angiogenesis, although some may promote angiogenesis indirectly (Mustonen and Alitalo, J. Cell Biol. 129:895–898, 1995).
One such receptor tyrosine kinase, known as fetal liver kinase 1 (flk-1), is a member of the type III subclass of RTKs. An alternative designation for human flk-1 is kinase insert domain-containing receptor (KDR) (Terman et al., Oncogene 6:1677–83, 1991), and the rat homolog has been termed TKr-C (Sarzani et al., Biochem. Biophys. Res. Comm. 186:706–714, 1992). DNAs encoding mouse, rat and human flk-1 have been isolated, and the nucleotide and encoded amino acid sequences reported (Matthews et al., Proc. Natl. Acad. Sci. USA, 88:9026–30, 1991; Terman et al., 1991, supra; Terman et al., Biochem. Biophys. Res. Comm. 187:1579–86, 1992; Sarzani et al., supra; and Millauer et al., Cell 72:835–846, 1993).
The type III subclass RTK designated fms-like tyrosine kinase-1 (flt-1) is related to flk-1 (DeVries et al., Science 255:989–991, 1992; Shibuya et al., Oncogene 5:519–524, 1990). Flt-1 is believed to be essential for endothelial organization during vascular development. Flt-1 expression is associated with early vascular development in mouse embryos, and with neovascularization during wound healing (Mustonen and Alitalo, supra). Expression of flt-1 in adult organs suggests an additional function for this receptor that is not related to cell growth (Mustonen and Alitalo, supra).
Another RTK that is related to flt1 and flk1 is flt4 (Galland et al., Oncogene 8:1233–40, 1993; Pajusola et al., Oncogene 8:2931–37, 1993). Features shared by these three receptors include the seven immunoglobulin-like domains in their extracellular region. The amino acid sequence of flt4 exhibits significant homology with the sequences of flt1 and flk-1, especially in the tyrosine kinase domain (Galland et al., supra). Unlike flt-1 and flk-1, however, a precursor form of flt-4 is cleaved during post-translational processing to form two disulfide-linked polypeptides (Pajusola et al., supra). Studies of Flt-4 expression during development support the theory of venous origin of lymphatic vessels (Kaipainen et al., Proc. Natl. Acad. Sci. USA 92:3566–70, April, 1995).
Given the crucial role of endothelial cells in angiogenesis, growth factors that act on endothelial cells are of particular interest for studies of the regulation of vascularization. One such factor is vascular endothelial cell growth factor (VEGF), which binds to both flk-1 and flt-1 with relatively high affinity and is mitogenic toward vascular endothelial cells (Terman et al., 1992, supra; Mustonen et al. supra; DeVries et al., supra). VEGF does not bind to flt4 (Pajusola et al., supra). The studies reported in Millauer et al., supra, suggest that VEGF and flk-1 are a ligand-receptor pair that play an important role in the formation and sprouting of blood vessels, termed vasculogenesis and angiogenesis, respectively.
Different forms of VEGF arising from alternative splicing of mRNA have been reported, including the four species described by Ferrara et al. (J. Cell. Biochem. 47:211–218, 1991). Both secreted and predominantly cell-associated species of VEGF were identified by Ferrara et al. supra, and the protein is known to exist in the form of disulfide linked dimers.
Placenta growth factor (PlGF) has an amino acid sequence that exhibits significant homology to the VEGF sequence (Park et al., J. Biol. Chem. 269:25646–54, 1994; Maglione et al. Oncogene 8:925–31, 1993). As with VEGF, different species of PlGF arise from alternative splicing of mRNA, and the protein exists in dimeric form (Park et al., supra). PlGF binds flt-1 with high affinity, but not flk-1 (Park et al., supra). PlGF potentiates the mitogenic effect of VEGF on endothelial cells when VEGF is present at low concentrations, but has no detectable effect when VEGF is present at higher concentrations (Park et al., supra.).
Studies of growth factors and receptors that are believed to regulate angiogenesis include those discussed above. Investigation into the existence and identity of other such receptors, and proteins that bind thereto, is desirable. Identifying such proteins would provide additional means for elucidating the effects of various ligand-receptor signaling systems on development and differentiation of the vascular system, as well as providing further insight into, and means for, regulation of such biological processes.
Inhibiting angiogenesis is desirable in certain clinical situations (e.g., to suppress growth and mestastasis of solid tumors, or in treating rheumatoid arthritis), whereas promoting vascularization is beneficial for treating other conditions (e.g., wound healing). Consequently, molecules that promote angiogenesis by transducing signals through the above-discussed receptors, and molecules capable of inhibiting such signal transduction, are both of interest.